PSCA: prostate stem cell antigen

ABSTRACT

The present invention provides methods for inhibiting the growth of prostate tumor cells expressing Prostate Stem Cell Antigen (PSCA), the methods comprising administering to a patient a monoclonal antibody which binds specifically to the extracellular domain of PSCA in an amount effective to inhibit growth of the prostate tumor cells.

This application is claiming the priority of provisional applications,U.S. Ser. No. 60/071,141 filed Jan. 12, 1998; U.S. Ser. No. 60/074,675,filed Feb. 13, 1998; which is a continuation of Ser. No. 08/814,279,filed Mar. 10, 1997.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Prostate cancer is currently the most common type of cancer in Americanmen and the second leading cause of cancer related death in thispopulation. In its advanced stages, prostate cancer metastasizespreferentially to bone, where it forms osteoblastic lesions. Afterinitial treatment with androgen ablation therapy, most metastaticprostate cancers become hormone-refractory and lethal. Currentdiagnostic and therapeutic modalities are limited by a lack ofspecificity and an inability to predict which patients are at risk ofdeveloping metastatic disease.

Most prostate cancers initially occur in the peripheral zone of theprostate gland, away from the urethra. Tumors within this zone may notproduce any symptoms and, as a result, most men with early-stageprostate cancer will not present clinical symptoms of the disease untilsignificant progression has occurred. Tumor progression into thetransition zone of the prostate may lead to urethral obstruction, thusproducing the first symptoms of the disease. However, these clinicalsymptoms are indistinguishable from the common non-malignant conditionof benign prostatic hyperplasia (BPH).

One of the fundamental problems in the diagnosis and treatment ofprostate cancer is the lack of a marker that can accurately detectearly-stage, localized tumors. Although a number of markers have beenidentified and some, like PSA, are in widespread clinical use, the idealprostate tumor marker has yet to be characterized. A similar problem isthe lack of an effective prognostic marker for determining which cancersare indolent and which ones are or will be aggressive. PSA, for example,fails to discriminate accurately between indolent and aggressivecancers. In addition, there is also a great need for markers which mightserve as ideal, prostate-specific targets for therapeutic methods suchas antibody-directed therapy, immunotherapy, and gene therapy.Currently, there is no effective treatment for the 20-40% of patientswho develop recurrent disease after surgery or radiation therapy or forthose patients who have metastatic disease at the time of diagnosis.Although hormone ablation therapy can palliate these patients, themajority inevitably progress to develop incurable, androgen-independentdisease (Lalani et al., 1997, Cancer Metastasis Rev. 16: 29-66).

Early detection and diagnosis of prostate cancer currently relies ondigital rectal examinations (DRE), prostate specific antigen (PSA)measurements, trausrectal ultrasonography (TRUS), and tansrectal needlebiopsy (TRNB). At present, serum PSA measurement in combination with DRErepresent the leading tool used to detect and diagnose prostate cancer.Both have major limitations which have fueled intensive research intofinding better diagnostic markers of this disease.

PSA is the most widely used tumor marker for screening, diagnosis, andmonitoring prostate cancer today. In particular, several immnunoassaysfor the detection of serum PSA are in widespread clinical use. Recently,a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSAmRNA in serum has been developed. However, PSA is not a disease-specificmarker, as elevated levels of PSA are detectable in large percentage ofpatients with BPH and prostatitis (25-86%)(Gao et al., 1997, Prostate31: 264-281), as well as in other nonmalignant disorders and in somenormal men, a factor which significantly limits the diagnosticspecificity of this marker. For example, elevations in serum PSA ofbetween 4 to 10 ng/ml are observed in BPH, and even higher values areobserved in prostatitis, particularly acute prostatitis. BPH is anextremely common condition in men. Further confusing the situation isthe fact that serun PSA elevations may be observed without anyindication of disease from DRE, and visa-versa. Moreover, it is nowrecognized that PSA is not prostate-specific (Gao et al., supra, forreview).

Various methods designed to improve the specificity of PSA-baseddetection have been described, such as measuring PSA density and theratio of free vs. complexed PSA. However, none of these methodologieshave been able to reproducibly distinguish benign from malignantprostate disease. In addition, PSA diagnostics have sensitivities ofbetween 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), andthus miss identifying prostate cancer in a significant population of menwith the disease.

Prostate -Specific Membrane Antigen (PSMA) is a recently described cellsurface marker of prostate cancer which has been the subject of variousstudies evaluating its use as a diagnostic and therapeutic marker. PSMAexpression is largely restricted to prostate tissues, but detectablelevels of PSMA MRNA have been observed in brain, salivary gland, smallintestine, and renal cell carcinoma (Israeli et al., 1993, Cancer Res53: 227-230). PSMA protein is highly expressed in most primary andmetastatic prostate cancers, but is also expressed in most normalintraepithelial neoplasia specimens (Gao et al., supra). Preliminaryresults using an Indium-111 labeled, anti-PSMA monoclonal antibody toimage recurrent prostate cancer show some promise (Sodee et al., 1996,Clin Nuc Med 21: 759-766). Whether PSMA will prove to be a usefultherapeutic target remains to be determined. However, PSMA is a hormonedependent antigen requiring the presence of functional androgenreceptor. Since not all prostate cancer cells express androgen receptor,PSMA's utility as a therapeutic target may be inherently limited.

Clinical staging of prostate cancer is another fundamental problemfacing urologists today. Currently, clinical staging relies on rectalexamination to determine whether the tumor remains within the borders ofthe prostatic capsule (locally confined) or extends beyond it (locallyadvanced), in combination with serum PSA determinations and transrectalultrasound guided biopsies. However, because of the subjectivityinvolved, clinical staging by DRE regularly underestimates oroverestimates local extension of the tumor, frequently misjudges itslocation, and correlates poorly with volume and extent of the tumor(Lee, C. T. and Osterling, J. E. Cancer of the Prostate: Diagnosis andStaging. In: Urologic Onclology, W. B. Saunders Company, Philadelphia,pp 357-377 (1997)). Serum PSA levels are also utilized for stagingpurposes, but PSA alone has not been able to reliably stage prostatetumors. No technique has proven reliable for predicting progression ofthe disease. Thus, there is a need for more reliable and informativestaging and prognostic methods in the management of prostate cancer.

SUMMARY OF THE INVENTION

The invention provides a novel prostate-specific cell-surface antigen,designated Prostate Stem Cell Antigen (PSCA), which is widelyover-expressed across all stages of prostate cancer, including highgrade prostatic intraepithelial neoplasia (PIN), androgen-dependent andandrogen-independent prostate tumors. The PSCA gene shows 30% homologyto stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly-6 family ofglycosylphosphatidylinositol (GPI)-anchored cell surface antigens, andencodes a 123 amino acid protein with an amino-terminal signal sequence,a carboxy-terminal GPI-anchoring sequence, and multiple N-glycosylationsites. PSCA mRNA expression is prostate specific in normal male tissuesand is highly upregulated in both androgen dependent and androgenindependent prostate cancer xenografts. In situ mRNA analysis localizesPSCA expression to the basal cell epithelium, the putative stem cellcompartment of the prostate. Flow cytometric analysis demonstrates thatPSCA is expressed predominantly on the cell surface and is anchored by aGPI linkage. Fluorescent in situ hybridization analysis localizes thePSCA gene to chromosome 8q24.2, a region of allelic gain in more than80% of prostate cancers.

PSCA may be an optimal therapeutic target in view of its cell surfacelocation, prostate specific expression, and greatly upregulatedexpression in prostate cancer cells. In this regard, the inventionprovides antibodies capable of binding to PSCA which can be usedtherapeutically to destroy prostate cancer cells. In addition, PSCAproteins and PSCA-encoding nucleic acid molecules may be used in variousimmunotherapeutic methods to promote immune-mediated destruction ofprostate tumors.

PSCA also may represent an ideal prostate cancer marker which can beused to discriminate between malignant prostate cancers, normal prostateglands and non-malignant neoplasias. For example, PSCA is expressed atvery high levels in prostate cancer in relation to benign prostatichyperplasia (BPH). In contrast, the widely used prostate cancer markerPSA is expressed at high levels in both normal prostate and BPH, but atlower levels in prostate cancer, rendering PSA expression useless fordistinguishing malignant prostate cancer from BPH or normal glands.Because PSCA expression is essentially the reverse of PSA expression,analysis of PSCA expression can be employed to distinguish prostatecancer from non-malignant conditions.

The genes encoding both human and murine PSCA have been isolated andtheir coding sequences elucidated and provided herein. Also provided arethe amino acid sequences of both human and murine PSCA. The inventionfurther provides various diagnostic assays for the detection,monitoring, and prognosis of prostate cancer, including nucleicacid-based and immunological assays. PSCA-specific monoclonal andpolyclonal antibodies and immunotherapeutic and other therapeuticmethods of treating prostate cancer are also provided. These and otheraspects of the invention are further described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Nucleotide sequences of a cDNA encoding human PSCA (ATCCDesignation 209612).

FIG. 1B. Translated amino acid sequences of a cDNA encoding human PSCA(ATCC Designation 209612).

FIG. 2. Nucleotide sequence of a cDNA encoding murine PSCA homologue(SEQ ID NO.:3) and amino acid sequence of murine PSCA (SEQ ID NO.:4).

FIG. 3. Alignment of amino acid sequences of human PSCA, murine PSCA,and human stem cell antigen-2 (hSCA-2). Shaded regions highlightconserved amino acids. Conserved cysteines are indicated by boldlettering. Four predicted N-glycosylation sites in PSCA are indicated byasterisks. The underlined amino acids at the beginning and end of theprotein represent N terminal hydrophobic signal sequences and C terminalGPI-anchoring sequences, respectively.

FIG. 4. Hydrophobicity plot of human PSCA.

FIG. 5. Chou-Fassman analysis of human PSCA.

FIG. 6. RT-PCR analysis of PSCA expression in various prostate cancercell lines and xenografts.

FIG. 7A. Restricted Expression of PSCA mRNA in normal and canceroustissues. RT-PCR analysis of PSCA expression in normal human tissuesdemonstrating high expression in prostate, placenta, and tonsils. 1 ngof reverse-inscribed first strand cDNA (Clontech, Palo Alto, Calif.)from the indicated tissues was amplified with PSCA gene specificprimers. Data shown are from 30 cycles of amplification.

FIG. 7B. Restricted Expression of PSCA mRNA in normal and canceroustissues. RT-PCR analysis of PSCA expression demonstrating high level inprostate cancer xenografts and normal tissue. 5 ng ofreverse-transcribed cDNA from the indicated tissues was amplified withPSCA gene specific primers. Amplification with β-actin gene specificprimers demonstrate normalization of the first strand cDNA of thevarious samples. Data shown are from 25 cycles of amplification. AD,androgen-dependent; Al, androgen-independent; IT, intratibial xenograft;C.L., cell line.

FIG. 8A. Schematic representation of human Thy-1/Ly-6 gene structures.

FIG. 8B. Schematic representation of murine PSCA gene structure.

FIG. 8C. Schematic representation of human PSCA gene structure.

FIG. 9A. Northern blot analysis of PSCA expression. Total RNA fromnormal prostate and LAPC-4 androgen dependent (AD) and independent (AI)prostate cancer xenografts were analyzed using PSCA or PSA specificprobes. Equivalent RNA loading and RNA integrity were demonstratedseparately by ethidium staining for 18S and 28S RNA.

FIG. 9B. Northern blot analysis of PSCA expression. Human multipletissue Northern blot analysis of PSCA. The filter was obtained fromClontech (Palo Alto, Calif.) and contains 2 ug of polyA RNA in eachlane.

FIG. 10-1. Northern blot analysis of PSCA expression in prostate cancerxenografts and tumor cell lines. PSCA demonstrates high level prostatecancer specific gene expression. 10 μg of total RNA from the indicatedtissues were size fractionated on an agarose/formaldehyde gel,transferred to nitrocellulose, and hybridized sequentially with³²P-labelled probes representing PSCA cDNA fragments. Shown are 4 hourand 72 hour autoradiogrphic exposures of the membrane. BPH, benignprostatic hyperplasia; AD, androgen-dependent; AI, androgen-independent;IT, intratibial xenograft; C.L., cell line.

FIG. 10-2. Northern blot analysis of PSM expression in prostate cancerxenografts and tumor cell lines. PSM demonstrates high level prostatecancer specific gene expression. 10 μg of total RNA from the indicatedtissues were size fractionated on an agarose/formaldehyde gel,transferred to nitrocellulose, and hybridized sequentially with³²P-labelled probes representing PSM cDNA fragments. Shown are 4 hourand 72 hour autoradiogrphic exposures of the membrane. BPH, benignprostatic hyperplasia; AD, androgen-dependent; AI, androgen-independent;IT, intratibial xenograft; C.L., cell line.

FIG. 10-3. Northern blot analysis of PSA expression in prostate cancerxenografts and tumor cell lines. 10 μg of total RNA from the indicatedtissues were size fractionated on an agarose/formaldehyde gel,transferred to nitrocellulose, and hybridized sequentially with³²P-labelled probes representing PSA cDNA fragments. Shown are 4 hourand 72 hour autoradiogrphic exposures of the membrane and the ethidiumbromide gel demonstrating equivalent loading of samples. BPH, benignprostatic hyperplasia; AD, androgen-dependent; AI, androgen-independent;IT, intratibial xenograft; C.L., cell line.

FIG. 11A. In situ hybridization with antisense riboprobe for human PSCAon normal prostate specimens. PSCA is expressed by a subset of basalcells within the basal cell epithelium, but not by the terminallydifferentiated secretory cells lining the prostatic ducts (400×magnification).

FIG. 11B. In situ hybridization with antisense riboprobe for human PSCAon normal and malignant prostate specimens. PSCA is expressed stronglyby a high grade prostatic intraepithelial neoplasia (PIN) and byinvasive prostate cancer glands, but is not detectable in normalepithelium at 40× magnification.

FIG. 11C. In situ hybridization with antisense riboprobe for human PSCAon malignant prostate specimens. Strong expression of PSCA in a case ofhigh grade carcinoma (200× magnification).

FIG. 12A. Biochemical analysis of PSCA. PSCA was immunoprecipitated from293T cells transiently transfected with a PSCA construct and thendigested with either N-glycosidase F or O-glycosidase, as described inMaterials and Methods.

FIG. 12B: Biochemical analysis of PSCA. PSCA was immunoprecipitated from293T transfected cells, as well as from conditioned media of these cells. Cell-associated PSCA migrates higher than secreted or shed PSCA on a15% polyacrylamide gel.

FIG. 12C. Biochemical analysis of PSCA. FACS analysis ofmock-transfected 293T cells, PSCA-transfected 293T cells and LAPC4prostate cancer xenograft cells using an affinity purified polyclonalanti-PSCA antibody. Cells were not permeabilized in order to detect onlysurface expression. The y axis represents relative cell number and the xaxis represents fluorescent staining intensity on a logarithmic scale.

FIG. 13. In situ hybridization of biotin-labeled PSCA probes to humanmetaphase cells from phytohemagglutinin-stimulated peripheral bloodlymphocytes. The chromosome 8 homologues are identified with arrows;specific labeling was observed at 8q24.2. The inset shows partialkaryotypes of two chromosome 8 homologues illustrating specific labelingat 8q24.2 (arrowheads). Images were obtained using a Zeiss Axiophotmicroscope coupled to a cooled charge coupled device (CCD) camera.Separate images of DAPI stained chromosomes and the hybridization signalwere merged using image analysis software (NU200 and Image 1.57).

FIG. 14A. Flow Cytometric analysis of cell surface PSCA expression onprostate cancer xenograft (LAPC-9) using anti-PSCA monoclonal antibodies1 G8 and 3E6, mouse anti-PSCA polyclonal serum, or control secondaryantibody. See Example 5 for details.

FIG. 14B. Flow Cytometric analysis of cell surface PSCA expression onprostate cancer cell line (LAPC-4) using anti-PSCA monoclonal antibodies1G8 and 3E6, mouse anti-PSCA polyclonal serum, or control secondaryantibody. See Example 5 for details.

FIG. 14C. Flow Cytometric analysis of cell surface PSCA expression onnormal prostate epithelial cells (PreC) using anti-PSCA monoclonalantibodies 1G8 and 3E6, mouse anti-PSCA polyclonal serum, or controlsecondary antibody. See Example 5 for details.

FIG. 15. Epitope mapping of anti-PSCA monoclonal antibodies conducted byWestern blot analysis of GST-PSCA fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Prostate Stem Cell Antigen (hereinafter“PSCA”). PSCA is a novel, glycosylphosphatidylinositol (GPI)-anchoredcell surface antigen which is expressed almost exclusively in prostatecells and which is overexpressed by both androgen-dependent andandrogen-independent prostate cancer cells. The expression of PSCA inprostate cancer may correlate with increasing grade.

PSCA mRNA is also expressed by a subset of basal cells in normalprostate. The basal cell epithelium is believed to contain theprogenitor cells for the terminally differentiated secretory cells(Borkhoff et al., 1994, Prostate 24: 114-118). Recent studies usingcytokeratin markers suggest that the basal cell epithelium contains atleast two distinct cellular subpopulations, one expressing cytokeratins5 and 14 and the other cytokeratins 5, 8 and 18 (Boichoff and Remberger,1996, Prostate 28: 98-106). The finding that PSCA is expressed by only asubset of basal cells suggests that PSCA may be a marker for one ofthese two basal cell lineages.

The biological function of PSCA is unknown. The Ly-6 gene family isinvolved in diverse cellular functions, including signal transductionand cell-cell adhesion. Signaling through SCA-2 has been demonstrated toprevent apoptosis in immature thymocytes (Noda et al., 1996, J. Exp.Med. 183: 2355-2360). Thy-1 is involved in T cell activation andtransmits signals through src-like tyrosine kinases (Thomas et al.,1992, J. Biol. Chem. 267: 12317-12322). Ly-6 genes have been implicatedboth in tumorigenesis and in homotypic cell adhesion (Bamezai and Rock,1995, Proc. Natl. Acad. Sci. USA 92: 4294-4298; Katz et al., 1994, Int.J. Cancer 59: 684-691; Brakenhoffet al., 1995, J. Cell Biol. 129:1677-1689). Based on its restricted expression in basal cells and itshomology to Sca-2, we hypothesize that PSCA may play a role instem/progenitor cell functions such as self-renewal (anti-apoptosis)and/or proliferation.

PSCA is highly conserved in mice and humans. The identification of aconserved gene which is predominantly restricted to prostate supportsthe hypothesis that PSCA may play an important role in normal prostatedevelopment.

In its various aspects, as described in detail below, the presentinvention provides PSCA proteins, antibodies, nucleic acid molecules,recombinant DNA molecules, transformed host cells, generation methods,assays, immunotherapeutic methods, transgenic animals, immunological andnucleic acid-based assays, and compositions.

PSCA PROTEINS

One aspect of the invention provides various PSCA proteins and peptidefragments thereof. As used herein, PSCA refers to a protein that has theamino acid sequence of human PSCA as provided in FIGS. 1B and 3, theamino acid sequence of the murine PSCA homologue as provided in FIG. 3,or the amino acid sequence of other mammalian PSCA homolognes, as wellas allelic variants and conservative substitution mutants of theseproteins that have PSCA activity. The PSCA proteins of the inventioninclude the specifically identified and characterized variants hereindescribed, as well as allelic variants, conservative substitutionvariants and homologs that can be isolated/generated and characterizedwithout undue experimentation following the methods outlined below. Forthe sake of convenience, all PSCA proteins will be collectively referredto as the PSCA proteins, the proteins of the invention, or PSCA.

The term “PSCA” includes all naturally occurring allelic variants,isoforms, and precursors of human PSCA as provided in FIGS. 1B and 3 andmurine PSCA as provided in FIG. 3. In general, for example, naturallyoccurring allelic variants of human PSCA will share significant homology(e.g., 70-90%) to the PSCA amino acid sequence provided in FIGS. 1B and3. Allelic variants, though possessing a slightly different amino acidsequence, may be expressed on the surface of prostate cells as a GPIlinked protein or may be secreted. Typically, allelic variants of thePSCA protein will contain conservative amino acid substitutions from thePSCA sequence herein described or will contain a substitution of anamino acid from a corresponding position in a PSCA homologue such as,for example, the murine PSCA homologue described herein.

One class of PSCA allelic variants will be proteins that share a highdegree of homology with at least a small region of the PSCA amino acidsequences presented in FIGS. 1B and 3, but will further contain aradical departure form the sequence, such as a non-conservativesubstitution, truncation, insertion or frame shift. Such alleles aretermed mutant alleles of PSCA and represent proteins that typically donot perform the same biological functions.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginie (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered‘conservative’ in particular environments.

The amino acid sequence of human PSCA protein is provided in FIGS. 1Band 3. Human PSCA is comprised of a single subunit of 123 amino acidsand contains an aminoterminal signal sequence, a carboxy-terminalGPI-anchoring sequence, and multiple N-glycosylation sites. PSCA shows30% homology to stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly6gene family, a group of cell surface proteins which mark the earliestphases of hematopoetic development The amino acid sequence of a murinePSCA homologue is shown in FIG. 3. Murine PSCA is a single subunitprotein of 123 amino acids having approximately 70% homology to humanPSCA and similar structural organization.

PSCA proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be isolated when physical,mechanical or chemical methods are employed to remove the PSCA proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated PSCA protein. A purified PSCA proteinmolecule will be substantially free of other proteins or molecules whichimpair the binding of PSCA to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of the PSCA protein include a purified PSCA protein and afunctional, soluble PSCA protein. One example of a functional solublePSCA protein has the amino acid sequence shown in FIG. 1B or a fragmentthereof. In one form, such functional, soluble PSCA proteins orfragments thereof retain the ability to bind antibody or other ligand.

The invention also provides peptides comprising biologically activefragments of the human and murine PSCA amino acid sequences shown inFIGS. 1B and 3. The peptides of the invention exhibit properties ofPSCA, such as the ability to elicit the generation of antibodies whichspecifically bind an epitope associated with PSCA. Such peptidefragments of the PSCA proteins can be generated using standard peptidesynthesis technology and the amino acid sequences of the human or murinePSCA proteins disclosed herein. Alternatively, recombinant methods canbe used to generate nucleic acid molecules that encode a fragment of thePSCA protein. In this regard, the PSCA-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of PSCA.As discussed below, peptide fragments of PSCA are particularly usefulin: generating domain specific antibodies; identifying agents that bindto PSCA or a PSCA domain; identifying cellular factors that bind to PSCAor a PSCA domain; and isolating homologs or other allelic forms of humanPSCA. PSCA peptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. As examples,hydrophobicity and Chou-Fasman plots of human PSCA are provided in FIGS.4 and 5, respectively. Fragments containing such residues areparticularly useful in generating subunit specific anti-PSCA antibodiesor in identifying cellular factors that bind to PSCA.

The PSCA proteins of the invention may be useful for a variety ofpurposes, including but not limited to their use as diagnostic and/orprognostic markers of prostate cancer and as targets for varioustherapeutic modalities, as further described below. PSCA proteins mayalso be used to identify and isolate ligands and other agents that bindto PSCA. In the normal prostate, PSCA is expressed exclusively in asubset of basal cells, suggesting that PSCA may be used as a marker fora specific cell lineage within basal epithelium. In addition,applicants' results suggest that this set of basal cells represent thetarget of neoplastic transformation. Accordingly for example,therapeutic strategies designed to eliminate or modulate the molecularfactors responsible for transformation may be specifically directed tothis population of cells via the PSCA cell surface protein.

PSCA ANTIBODIES

The invention further provides antibodies that bind to PSCA. The mostpreferred antibodies will selectively bind to PSCA and will not bind (orwill bind weakly) to non-PSCA proteins. Anti-PSCA antibodies that areparticularly contemplated include monoclonal and polyclonal antibodiesas well as fragments containing the antigen binding domain and/or one ormore complement determining regions of these antibodies.

In one embodiment, the PSCA antibodies specifically bind to theextracellular domain of a PSCA protein. In other embodiments, the PSCAantibodies specifically bind to other domains of a PSCA protein orprecursor. As will be understood by those skilled in the art, theregions or epitopes of a PSCA protein to which an antibody is directedmay vary with the intended application. For example, antibodies intendedfor use in an immunoassay for the detection of membrane-bound PSCA onviable prostate cancer cells should be directed to an accessible epitopeon membrane-bound PSCA. Examples of such antibodies are described theExamples which follow. Antibodies which recognize other epitopes may beuseful for the identification of PSCA within damaged or dying cells, forthe detection of secreted PSCA proteins or fragments thereof. Theinvention also encompasses antibody fragments which specificallyrecognize an PSCA protein. As used herein, an antibody fragment isdefined as at least a portion of the variable region of theimmunoglobulin molecule which binds to its target, i.e., the antigenbinding region. Some of the constant region of the immunoglobulin may beincluded.

The prostate-specificity of PSCA, its overexpression in bothandrogen-dependent and androgen-independent prostate cancer cells, andthe cell surface location of PSCA represent characteristics of anexcellent marker for screening, diagnosis, prognosis, and follow-upassays and imaging methods. In addition, these characteristics indicatethat PSCA may be an excellent target for therapeutic methods such astargeted antibody therapy, immunotherapy, and gene therapy.

PSCA antibodies of the invention may be particularly useful indiagnostic assays, imaging methodologies, and therapeutic methods in themanagement of prostate cancer. The invention provides variousimmunological assays useful for the detection of PSCA proteins and forthe diagnosis of prostate cancer. Such assays generally comprise one ormore PSCA antibodies capable of recognizing and binding a PSCA protein,and include various immunological assay formats well known in the art,including but not limited to various types of radioimmunoassays,enzyme-lined immunosorbent assays ELISA), enzyme-linkedimmunofluorescent assays (ELIFA), and the like. In addition,immunological imaging methods capable of detecting prostate cancer arealso provided by the invention, including but limited toradioscintigraphic imaging methods using labeled PSCA antibodies. Suchassays may be clinically useful in the detection, monitoring, andprognosis of prostate cancer.

In one embodiment, PSCA antibodies and fragments thereof are used fordetecting the presence of a prostate cancer, PIN, or basal epithelialcell expressing a PSCA protein. The presence of such PSCA+cells withinvarious biological samples, including serum, prostate and other tissuebiopsy specimens, other tissues such as bone, urine, etc., may bedetected with PSCA antibodies. In addition, PSCA antibodies may be usedin various imaging methodologies, such as immunoscintigraphy withInduim-111 (or other isotope) conjugated antibody. For example, animaging protocol similar to the one recently described using an In-111conjugated anti-PSMA antibody may be used to detect recurrent andmetastatic prostate carcinomas (Sodee et al., 1997, Clin Nuc Med 21:759-766). In relation to other markers of prostate cancer, such as PSMAfor example, PSCA may be particularly useful for targeting androgenreceptor-negative prostate cancer cells. PSCA antibodies may also beused therapeutically to inhibit PSCA function.

PSCA antibodies may also be used in methods for purifying PSCA proteinsand peptides and for isolating PSCA homologues and related molecules.For example, in one embodiment, the method of purifying a PSCA proteincomprises incubating a PSCA antibody, which has been coupled to a solidmatrix, with a lysate or other solution containing PSCA under conditionswhich permit the PSCA antibody to bind to PSCA; washing the solid matrixto eliminate impurities; and eluting the PSCA from the coupled antibody.

Additionally, PSCA antibodies may be used to isolate PSCA positive cellsusing cell sorting and purification techniques. The presence of PSCA onprostate tumor cells may be used to distinguish and isolate humanprostate cancer cells from other cells. In particular, PSCA antibodiesmay be used to isolate prostate cancer cells from xenograft tumortissue, from cells in culture, etc., using antibody-based cell sortingor affinity purification techniques. Other uses of the PSCA antibodiesof the invention include generating anti-idiotypic antibodies that nimicthe PSCA protein.

The ability to generate large quantities of relatively pure humanprostate cancer cells which can be grown in tissue culture or asxenograft tumors in animal models (e.g., SCID or other immune deficientmice) provides many advantages, including, for example, permitting theevaluation of various transgenes or candidate therapeutic compounds onthe growth or other phenotypic characteristics of a relativelyhomogeneous population of prostate cancer cells. Additionally, thisfeature of the invention also permits the isolation of highly enrichedpreparations of human prostate cancer specific nucleic acids inquantities sufficient for various molecular manipulations. For example,large quantities of such nucleic acid preparations will assist in theidentification of rare genes with biological relevance to prostatecancer disease progression.

Another valuable application of this aspect of the invention is theability to analyze and experiment with relatively pure preparations ofviable prostate tumor cells cloned from individual patients with locallyadvanced or metastatic disease. In this way, for example, an individualpatient's prostate cancer cells may be expanded from a limited biopsysample and then tested for the presence of diagnostic and prognosticgenes, proteins, chromosomal aberrations, gene expression profiles, orother relevant genotypic and phenotypic characteristics, without thepotentially confounding variable of contaminating cells. In addition,such cells may be evaluated for neoplastic aggressiveness and metastaticpotential in animal models. Similarly, patient-specific prostate cancervaccines and cellular immunotherapeutics may be created from such cellpreparations.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a PSCA protein, peptide, or fragment, in isolatedor immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press,NY (1989)). In addition, fusion proteins of PSCA may also be used, suchas a PSCA GST-fusion protein. Cells expressing or overexpressing PSCAmay also be used for immunizations. Similarly, any cell engineered toexpress PSCA may be used. This strategy may result in the production ofmonoclonal antibodies with enhanced capacities for recognizingendogenous PSCA. For example, using standard technologies described inExample 5 and standard hybridoma protocols (Harlow and Lane, 1988,Antibodies: A Laboratory Manual. (Cold Spring Harbor Press)), hybridomasproducing a monoclonal antibody designated 1G8, 2H9, 3C5, and 4A10 w=egenerated. The hybridomas of the present invention were deposited withthe Patent Culture Depository of the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA. Thedeposits were granted the following ATCC Accession Numbers: HB12612(1G8), HB-12614 (2H9), HB-12616 (3CS), and HB-12617 (4A10). The depositswere granted the date of Dec. 11, 1998.

The amino acid sequence of PSCA presented herein may be used to selectspecific regions of the PSCA protein for generating antibodies. Forexample, hydrophobicity and hydrophobicity analyses of the PSCA aminoacid sequence may be used to identify hydrophilic regions in the PSCAstructure. Regions of the PSCA protein that show immunogenic structure,as well as other regions and domains, can readily be identified usingvarious other methods known in the ark such as Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis. Fragments containing these residues areparticularly suited in generating specific classes of anti-PSCAantibodies. Particularly useful fragments include, but are not limitedto, the sequences TARIRAVGLLTVISK (SEQ ID NO:8) and VDDSQDYYVGKK (SEQ IDNO:9). As described in Example 2, below, a rabbit polyclonal antibodywas generated against the former fragment, prepared as a syntheticpeptide, and affinity purified using a PSCA-glutathione S transferasefusion protein. Recognition of PSCA by this antibody was established byimmunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. This antibody alsoidentified the cell surface expression of PSCA in PSCA-transfected 293Tand LAPC-4 cells using fluorescence activated cell sorting (FACS).

Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation using, for example, carbodilmidereagents may be used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., may be effective.Administration of a PSCA immunogen is conducted generally by injectionover a suitable time period and with use of a suitable adjuvant, as isgenerally understood in the art During the immunization schedule, titersof antibodies can be taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, monoclonalantibody preparations are preferred. Immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the PSCA protein or PSCAfragment When the appropriate immortalized cell culture secreting thedesired antibody is identified, the cells can be cultured either invitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

The generation of two monoclonal antibodies (MAbs) capable of binding tocell surface PSCA is described in Example 5. Epitope mapping of theseMAbs indicates that they recognize different epitopes on the PSCAprotein, one recognizing an epitope within the carboy-terminal regionand the other recognizing an epitope within the amino-terminal regionSuch PSCA antibodies may be particularly well suited to use in asandwich-formatted ELISA, given their differing epitope bindingcharacteristics.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the PSCA protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.

The antibody or fragment thereof of the invention may be labeled with adetectable marker or conjugated to a second molecule, such as acytotoxic agent, and used for targeting the second molecule to an PSCApositive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, inDeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice ofOncology, 4th ed., J. B. Lippincott Co., Philadelphia, 2624-2636).Examples of cytotoxic agents include, but are not limited to ricin,doxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin(PE) A, PE40, ricin, abrin, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes. Suitable detectablemarkers include, but are not limited to, a radioisotope, a fluorescentcompound, a bioluminescent compound, chemiluminescent compound, a metalchelator or an enzyme.

PSCA antibodies may be used systemically to treat prostate cancer. PSCAantibodies conjugated with toxic agents, such as ricin, as well asunconjugated antibodies may be useful therapeutic agents naturallytargeted to PSCA-bearing prostate cancer cells. Techniques forconjugating therapeutic agents to antibodies are well known (see, e.g.,Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldet al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Helistrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications.Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982)). The use of PSCA antibodies astherapeutic agents is further described in the subsection “PROSTATECANCER IMMUNOTHERAPY” below.

PSCA-ENCODING NUCLEIC ACID MOLECULES

Another aspect of the invention provides various nucleic acid moleculesencoding PSCA proteins and fragments thereof, preferably in isolatedform, including DNA, RNA, DNA/RNA hybrid, and related molecules, nucleicacid molecules complementary to the PSCA coding sequence or a partthereof, and those which hybridize to the PSCA gene or to PSCA-encodingnucleic acids. Particularly preferred nucleic acid molecules will have anucleotide sequence substantial identical to or complementary to thehuman or murine DNA sequences herein disclosed. Specificallycontemplated are genomic DNA, cDNAs, ribozymes, and antisense molecules,as well as nucleic acids based on an alternative backbone or includingalternative bases, whether derived from natural sources or synthesized.For example, antisense molecules can be RNAs or other molecules,including peptide nucleic acids (PNAs) or non-nucleic acid moleculessuch as phosphorothioate derivatives, that specifically bind DNA or RNAin a base pair-dependent manner. A skilled artisan can readily obtainthese classes of nucleic acid molecules using the herein described PSCAsequences. For convenience, PSCA-encoding nucleic acid molecules will bereferred to herein as PSCA-encoding nucleic acid molecules, PSCA genes,or PSCA sequences.

The nucleotide sequence of a cDNA encoding one allelic form of humanPSCA is provided in FIG. 1A. The nucleotide sequence of a cDNA encodinga murine PSCA homologue (“murine PSCA”) is provided in FIG. 2. Genoricclones of human and murine PSCA have also been isolated, as described inExample 4. Both the human and murine genomic clones contain three exonsencoding the translated and 3′ untranslated regions of the PSCA gene. Afourth exon encoding a 5′ untranslated region is presumed to exist basedon PSCA's homology to other members of the Ly-6 and Thy-1 gene families(FIG. 8).

The human PSCA gene maps to chromosome 8q24.2. Human stem cell antigen 2(RIG-E), as well as one other recently identified human Ly-6 homologueE48) are also localized to this region, suggesting that a large familyof related genes may exist at this locus (Brakenhoff et al., 1995,supra; Mao et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5910-5914).Intriguingly, chromosome gq has been reported to be a region of allelicgain and amplification in a majority of advanced and recurrent prostatecancers (Cher et al., 1994, Genes Chrom. Cancer 11: 153-162). c-myclocalizes proximal to PSCA at chromosome 8q24 and extra copies of c-myc(either through allelic gain or amplification) have been found in 68% ofprimary prostate tumors and 96% of metastases (Jenkins et al., 1997,Cancer Res. 57: 524-531).

Embodiments of the PSCA-encoding nucleic acid molecules of the inventioninclude primers, which allow the specific amplification of nucleic acidmolecules of the invention or of any specific parts thereof, and probesthat selectively or specifically hybridize to nucleic acid molecules ofthe invention or to any part thereof. The nucleic acid probes can belabeled with a detectable marker. Examples of a detectable markerinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme. Such labeled probes can be used to diagnosis the presenceof a PSCA protein as a means for diagnosing cell expressing a PSCAprotein. Technologies for generating DNA and RNA probes are well known.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules that encode polypeptides other than PSCA. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated PSCA-encoding nucleic acid molecule.

The invention further provides fragments of the PSCA-encoding nucleicacid molecules of the present invention. As used herein, a fragment of aPSCA-encoding nucleic acid molecule refers to a small portion of theentire PSCA-encoding sequence. The size of the fragment will bedetermined by its intended use. For example, if the fragment is chosenso as to encode an active portion of the PSCA protein, such an activedomain, effector binding site or GPI binding domain, then the fragmentwill need to be large enough to encode the functional region(s) of thePSCA protein. If the fragment is to be used as a nucleic acid probe orPCR primer, then the fragment length is chosen so as to obtain arelatively small number of false positives during probing/priming.Fragments of human PSCA that are particularly useful as selectivehybridization probes or PCR primers can be readily identified from theentire PSCA sequence using art-known methods. One set of PCR primersthat are used for RT-PCR analysis comprise 5′- TGCTTGCCCTGTTGATGGCAG(SEQ ID NO:12) and 3′-CCAGAGCAGCAGGCCGAGTGCA (SEQ ID NO:13).

Another class of fragments of PSCA-encoding nucleic acid molecules arethe expression control sequence found upstream and downstream from thePSCA-encoding region found in genomic clones of the PSCA gene.Specifically, prostate specific expression control elements can beidentified as being 5′ to the PSCA-encoding region found in genomicclones of the PSCA gene. Such expression control sequence are useful ingenerating expression vectors for expressing genes in a prostatespecific fashion. A skilled artisan can readily use the PSCA cDNAsequence herein described to isolate and identify genomic PSCA sequencesand the expression control elements found in the PSCA gene.

METHODS FOR ISOLATING OTHER PSCA-ENCODING NUCLEIC ACID MOLECULES

The PSCA-encoding nucleic acid molecules described herein enable theisolation of PSCA homologues, alternatively sliced isoforms, allelicvariants, and mutant forms of the PSCA protein as well as their codingand gene sequences. The most preferred source of PSCA homologs aremammalian organisms.

For example, a portion of the PSCA-encoding sequence herein describedcan be synthesized and used as a probe to retrieve DNA encoding a memberof the PSCA family of proteins from organisms other than human, allelicvariants of the human PSCA protein herein described, and genomicsequence containing the PSCA gene. Oligomers containing approximately18-20 nucleotides (encoding about a 6-7 amino acid stretch) are preparedand used to screen genomic DNA or cDNA libraries to obtain hybridizationunder stringent conditions or conditions of sufficient stringency toeliminate an undue level of false positives. In a particular embodiment,cDNA encoding human PSCA was used to isolate a full length cDNA encodingthe murine PSCA homologue as described in Example 3 herein. The murineclone encodes a protein with 70% amino acid identity to human PSCA.

In addition, the amino acid sequence of the human PSCA protein may beused to generate antibody probes to screen expression libraries preparedfrom cells. Typically, polyclonal antiserum from mammals such as rabbitsimmunized with the purified protein (as described below) or monoclonalantibodies can be used to probe an expression library, prepared from atarget organism, to obtain the appropriate coding sequence for a PSCAhomologue. The cloned cDNA sequence can be expressed as a fusionprotein, expressed directly using its own control sequences, orexpressed by constructing an expression cassette using control sequencesappropriate to the particular host used for expression of the enzyme.Genomic clones containing PSCA genes may be obtained using molecularcloning methods well known in the art. In one embodiment, a humangenomic clone of approximately 14 kb containing exons 1-4 of the PSCAgene was obtained by screening a lambda phage library with a human PSCAcDNA probe, as more completely described in Example 4 herein. In anotherembodiment, a genomic clone of approximately 10 kb containing the murinePSCA gene was obtained by screening a murine BAC (bacterial artificialchromosome) library with a murine PSCA cDNA (also described in Example4).

Additionally, pairs of oligonucleotide primers can be prepared for usein a polymerase chain reaction (PCR) to selectively amplify/clone aPSCA-encoding nucleic acid molecule, or fragment thereof A PCRdenature/anneal/extend cycle for using such PCR primers is well known inthe art and can readily be adapted for use in isolating otherPSCA-encoding nucleic acid molecules. Regions of the human PSCA genethat are particularly well suited for use as a probe or as primers canbe readily identified.

Non-human homologues of PSCA, naturally occurring allelic variants ofPSCA and genomic PSCA sequences will share a high degree of homology tothe human PSCA sequences herein described. In general, such nucleic acidmolecules will hybridize to the human PSCA sequence under stringentconditions. Such sequences will typically contain at least 70% homology,preferably at least 80%, most preferably at least 90% homology to thehuman PSCA sequence. “Stringent conditions” are those that (1) employlow ionic strength and high temperature for washing, for example, 0.015MNaCl/0.0015M sodium titrate/0.1% SDS at 50EC., or (2) employ duringhybridization a denaturing agent such as formamide, for example, 500%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42EC. Another example is use of 50%formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 Tg/ml), 0.1% SDS, and 10% dextan sulfateat 42EC., with washes at 42EC. in 0.2×SSC and 0.1% SDS. A skilledartisan can readily determine and vary the stringency conditionsappropriately to obtain a clear and detectable hybridization signal.

RECOMBINANT DNA MOLECULES CONTAINING PSCA-ENCODING NUCLEIC ACIDS

Also provided are recombinant DNA molecules (rDNAs) that contain aPSCA-encoding sequences as herein described, or a fragment thereof. Asused herein, a rDNA molecule is a DNA molecule that has been subjectedto molecular manipulation in vitro. Methods for generating rDNAmolecules are well known in the art for example, see Sambrook et al.,Molecular Cloning (1989). In the preferred rDNA molecules of the presentinvention, a PSCA-encoding DNA sequence that encodes a PSCA protein or afragment of PSCA, is operably linked to one or more expression controlsequences and/or vector sequences. The rDNA molecule can encode eitherthe entire PSCA protein or can encode a fragment of the PSCA protein.

The choice of vector and/or expression control sequences to which thePSCA-encoding sequence is operably linked depends directly, as is wellknown in the art, on the functional properties desired, e.g., proteinexpression, and the host cell to be transformed. A vector contemplatedby the present invention is at least capable of directing thereplication or insertion into the host chromosome, and preferably alsoexpression, of the PSCA-encoding sequence included in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, enhancers, transcription terminators andother regulatory elements. Preferably, an inducible promoter that isreadily controlled, such as being responsive to a nutrient in the hostcell's medium, is used.

In one embodiment, the vector containing a PSCA-encoding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule intrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the PSCA-encoding sequence in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Various viral vectors well known to thoseskilled in the art may also be used, such as, for example, a number ofwell known retroviral vectors.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to variant rDNAmolecules that contain a PSCA-encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDTI (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. Southern et al., J Mol Anal Genet (1982)1:327-341.Alternatively, the selectable marker can be present on a separateplasmid, and the two vectors are introduced by cotransfection of thehost cell, and selected by culturing in the presence of the appropriatedrug for the selectable marker.

In accordance with the practice of the invention, the vector can be aplasmid, cosmid or phage vector encoding the cDNA molecule discussedabove. Additionally, the invention provides a host-vector systemcomprising the plasmid, cosmid or phage vector transfected into asuitable eucaryotic host cell. Examples of suitable eucaryotic hostcells include a yeast cell, a plant cell, or an animal cell, such as amammalian cell. Examples of suitable cells include the LnCaP, LAPC4, andother prostate cancer cell lines. The host-vector system is useful forthe production of an PSCA protein. Alternatively, the host cell can beprokaryotic, such as a bacterial cell.

TRANSFORMED HOST CELLS

The invention further provides host cells transformed with a nucleicacid molecule that encodes a PSCA protein or a fragment thereof. Thehost cell can be either prokaryotic or eukaryotic. Eukaryotic cellsuseful for expression of a PSCA protein are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of a PSCA gene.Preferred eukaryotic host cells include, but are not limited to, yeast,insect and mammalian cells, preferably vertebrate cells such as thosefrom a mouse, rat, monkey or human fibroblastic cell line. Prostatecancer cell lines, such as the LnCaP and LAPC-4 cell lines may also beused. Any prokaryotic host can be used to express a PSCA-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with an rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen etal., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). With regard to transformation of vertebrate cellswith vectors containing rDNAs, electroporation, cationic lipid or salttreatment methods are typically employed, see, for example, Graham etal., Virol (1973) 52:456; Wigler et al., Proc Nat Acad Sci USA (1979)76:1373-76.

Successfully transformed cells, i.e., cells that contain an rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

RECOMBINANT METHODS OF GENERATING PSCA PROTEINS

The invention further provides methods for producing a PSCA proteinusing one of the PSCA-encoding nucleic acid molecules herein described.In general terms, the production of a recombinant PSCA protein typicallyinvolves the following steps.

First, a nucleic acid molecule is obtained that encodes a PSCA proteinor a fragment thereof, such as the nucleic acid molecule depicted inFIG. 1A. The PSCA-encoding nucleic acid molecule is then preferablyplaced in an operable linkage with suitable control sequences, asdescribed above, to generate an expression unit containing thePSCA-encoding sequence. The expression unit is used to transform asuitable host and the transformed host is cultured under conditions thatallow the production of the PSCA protein. Optionally the PSCA protein isisolated from the medium or from the cells; recovery and purification ofthe protein may not be necessary in some instances where some impuritiesmay be tolerated.

Each of the foregoing steps may be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in an appropriate host. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using an appropriate combination of replicons and controlsequences. The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the geneand were discussed in detail earlier. Suitable restriction sites can, ifnot normally available, be added to the ends of the coding sequence soas to provide an excisable gene to insert into these vectors. A skilledartisan can readily adapt any host/expression system known in the artfor use with PSCA-encoding sequences to produce a PSCA protein.

ASSAYS FOR IDENTIFYING PSCA LIGANDS AND OTHER BINDING AGENTS

Another aspect of the invention relates to assays and methods which canbe used to identify PSCA ligands and other agents and cellularconstituents that bind to PSCA. Specifically, PSCA ligands and otheragents and cellular constituents that bind to PSCA can be identified bythe ability of the PSCA ligand or other agent or constituent to bind toPSCA and/or the ability to inhibit/stimulate PSCA activity. Assays forPSCA activity (e.g., binding) using a PSCA protein are suitable for usein high through-put screening methods.

In one embodiment, the assay comprises mixing PSCA with a test agent orcellular extract After mixing under conditions that allow association ofPSCA with the agent or component of the extract, the mixture is analyzedto determine if the agent/component is bound to PSCA. Bindingagents/components are identified as being able to bind to PSCA.Alternatively or consecutively, PSCA activity can be directly assessedas a means for identifying agonists and antagonists of PSCA activity.

Alternatively, targets that bind to a PSCA protein can be identifiedusing a yeast two-hybrid system or using a binding-capture assay. In theyeast two hybrid system, an expression unit encoding a fusion proteinmade up of one subunit of a two subunit transcription factor and thePSCA protein is introduced and expressed in a yeast cell. The cell isfurther modified to contain (1) an expression unit encoding a detectablemarker whose expression requires the two subunit transcription factorfor expression and (2) an expression unit that encodes a fusion proteinmade up of the second subunit of the transcription factor and a clonedsegment of DNA. If the cloned segment of DNA encodes a protein thatbinds to the PSCA protein, the expression results in the interaction ofthe PSCA and the encoded protein. This brings the two subunits of thetranscription factor into binding proximity, allowing reconstitution ofthe transcription factor. This results in the expression of thedetectable marker. The yeast two hybrid system is particularly useful inscreening a library of cDNA encoding segments for cellular bindingpartners of PSCA.

PSCA proteins which may be used in the above assays include, but are notlimited to, an isolated PSCA protein, a fragment of a PSCA protein, acell that has been altered to express a PSCA protein, or a fraction of acell that has been altered to express a PSCA protein. Further, the PSCAprotein can be the entire PSCA protein or a defined fragment of the PSCAprotein. It will be apparent to one of ordinary skill in the art that solong as the PSCA protein can be assayed for agent binding, e.g., by ashift in molecular weight or activity, the present assay can be used.

The method used to identify whether an agent/cellular component binds toa PSCA protein will be based primarily on the nature of the PSCA proteinused. For example, a gel retardation assay can be used to determinewhether an agent binds to PSCA or a fragment thereof. Alternatively,immunodetection and biochip technologies can be adopted for use with thePSCA protein. A skilled artisan can readily employ numerous art-knowntechniques for determining whether a particular agent binds to a PSCAprotein.

Agents and cellular components can be further tested for the ability tomodulate the activity of a PSCA protein using a cell-free assay systemor a cellular assay system. As the activities of the PSCA protein becomemore defined, functional assays based on the identified activity can beemployed.

As used herein, an agent is said to antagonize PSCA activity when theagent reduces PSCA activity. The preferred antagonist will selectivelyantagonize PSCA, not affecting any other cellular proteins. Further, thepreferred antagonist will reduce PSCA activity by more than 50%, morepreferably by more than 90%, most preferably eliminating all PSCAactivity.

As used herein, an agent is said to agonize PSCA activity when the agentincreases PSCA activity. The preferred agonist will selectively agonizePSCA, not affecting any other cellular proteins. Further, the preferredantagonist will increase PSCA activity by more than 50%, more preferablyby more than 90%, most preferably more than doubling PSCA activity.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences of the PSCA protein. An example of randomlyselected agents is the use of a chemical library or a peptidecombinatorial library, or a growth broth of an organism or plantextract.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that make up the PSCAprotein. For example, a rationally selected peptide agent can be apeptide whose amino acid sequence is identical to a fragment of a PSCAprotein.

The agents tested in the methods of the present invention can be, asexamples, peptides, small molecules, and vitamin derivatives, as well ascarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents used in the presentscreening method. One class of agents of the present invention arepeptide agents whose amino acid sequences are chosen based on the aminoacid sequence of the PSCA protein. Small peptide agents can serve ascompetitive inhibitors of PSCA protein assembly.

Peptide agents can be prepared using standard solid phase (or solutionphase) peptide synthesis methods, as is known in the art In addition,the DNA encoding these peptides may be synthesized using commerciallyavailable oligonucleotide synthesis instrumentation and producedrecombinantly using standard recombinant production systems. Theproduction using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the PSCA protein. As describedabove, antibodies are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the PSCA protein intended to be targeted by the antibodies. Criticalregions may include the domains identified in FIGS. 4 and 5. Such agentscan be used in competitive binding studies to identify second generationinhibitory agents.

The cellular extracts tested in the methods of the present invention canbe, as examples, aqueous extracts of cells or tissues, organic extractsof cells or tissues or partially purified cellular fractions. A skilledartisan can readily recognize that there is no limit as to the source ofthe cellular extract used in the screening method of the presentinvention.

Agents that bind a PSCA protein, such as a PSCA antibody, can be used tomodulate the activity of PSCA, to target anticancer agents toappropriate mammalian cells, or to identify agents that block theinteraction with PSCA. Cells expressing PSCA can be targeted oridentified by using an agent that binds to PSCA.

How the PSCA binding agents will be used depends on the nature of thePSCA binding agent. For example, a PSCA binding agent can be used to:deliver conjugated toxins, such a diphtheria toxin, cholera toxin, ricinor pseudomonas exotoxin, to a PSCA expressing cell; modulate PSCAactivity; to directly kill PSCA expressing cells; or in screens toidentify competitive binding agents. For example, a PSCA inhibitoryagent can be used to directly inhibit the growth of PSCA expressingcells whereas a PSCA binding agent can be used as a diagnostic agent.

PROSTATE CANCER IMMUNOTHERAPY

The invention provides various immunotherapeutic methods for treatingprostate cancer, including antibody therapy, in vivo vaccines, and exvivo immunotherapy approaches. In one approach, the invention providesPSCA antibodies which may be used systemically to treat prostate cancer.For example, unconjugated PSCA antibody may be introduced into a patientsuch that the antibody binds to PSCA on prostate cancer cells anmediates the destruction of the cells, and the tumor, by mechanismswhich may include complement-mediated cytolysis, antibody-dependentcellular cytotoxicity, altering the physiologic function of PSCA, and/orthe inhibition of ligand binding or signal transduction pathways. PSCAantibodies conjugated to toxic agents such as ricin may also be usedtherapeutically to deliver the toxic agent directly to PSCA-bearingprostate tumor cells and thereby destroy the tumor.

Prostate cancer immunotherapy using PSCA antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit Rev Immunol 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186;Tsunenari et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyket al., 1992, Cancer Res 52: 2771-2776), B-cell lymphoma (Funakosbi etal., 1996, J Immunother Emphasis Tumor Immunol 19: 93-101), leukemia(Thong et al., 1996, Leuk Res 20: 581-589), colorectal cancer (Noun etal., 1994, Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res55: 4398-4403), and breast cancer (Shepard et al., 1991, J Clin Immunol11: 117-127).

The invention further provides vaccines formulated to contain a PSCAprotein or fragment thereof The use of a tumor antigen in a vaccine forgenerating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159:3113-3117). Such methods can be readily practiced by employing a PSCAprotein, or fragment thereof, or a PSCA-encoding nucleic acid moleculeand recombinant vectors capable of expressing and appropriatelypresenting the PSCA immunogen.

For example, viral gene delivery systems may be used to deliver aPSCA-encoding nucleic acid molecule. Various viral gene delivery systemswhich can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a PSCA protein or fragment thereof introduced into the patient(e.g., intrumuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human PSCA cDNA may be employed. In anotherembodiment, PSCA nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a PSCA protein which are capable of optimallybinding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present PSCA antigen to a patient's immunesystem. Dendritic cells express MHC class I and II, B7 costimulator, andIL-12, and are thus highly specialized antigen presenting cells. Inprostate cancer, autologous dendritic cells pulsed with peptides of theprostate-specific membrane antigen (PSMA) are being used in a Phase Iclinical trial to stimulate prostate cancer patients' immune systems(Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate29: 371-380). Dendritic cells can be used to present PSCA peptides to Tcells in the context of MHC class I and II molecules. In one embodiment,autologous dendritic cells are pulsed with PSCA peptides capable ofbinding to MHC molecules. In another embodiment, dendritic cells arepulsed with the complete PSCA protein. Yet another embodiment involvesengineering the overexpression of the PSCA gene in dendritic cells usingvarious implementing vectors known in the art, such as adenovirus(Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Hendersonet al., 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associatedvirus, DNA transfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869),and tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186: 1177-1182).

Anti-idiotypic anti-PSCA antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga PSCA protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-PSCA antibodies that mimic an epitope on aPSCA protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in anti-idiotypic therapy as presently practicedwith other anti-idiotypic antibodies directed against tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing PSCA. Using the PSCA-encoding DNA moleculesdescribed herein, constructs comprising DNA encoding a PSCAprotein/imunogen and appropriate regulatory sequences may be injecteddirectly into muscle or skin of an individual, such that the cells ofthe muscle or skin take-up the construct and express the encoded PSCAprotein/immunogen. The PSCA protein/immunogen may be expressed as a cellsurface protein or be secreted. Expression of the PSCA protein/immunogenresults in the generation, of prophylactic or therapeutic humoral andcellular immunity against prostate cancer. Various prophylactic andtherapeutic genetic immunization techniques known in the art may be used(for review, see information and references published at internetaddress www.genweb.com).

METHODS FOR IDENTIFYING PSCA PROTEINS AND PSCA GENES AND RNA

The invention provides methods for identifying cells, tissues ororganisms expressing a PSCA protein or a PSCA gene. Such methods can beused to diagnose the presence of cells or an organism that expresses aPSCA protein in vivo or in vitro. The methods of the present inventionare particularly useful in the determining the presence of cells thatmediate pathological conditions of the prostate. Specifically, thepresence of a PSCA protein can be identified by determining whether aPSCA protein, or nucleic acid encoding a PSCA protein, is expressed. Theexpression of a PSCA protein can be used as a means for diagnosing thepresence of cells, tissues or an organism that expresses a PSCA proteinor gene.

A variety of immunological and molecular genetic techniques can be usedto determine if a PSCA protein is expressed/produced in a particularcell or sample. In general, an extract containing nucleic acid moleculesor an extract containing proteins is prepared. The extract is thenassayed to determine whether a PSCA protein, or a PSCA-encoding nucleicacid molecule, is produced in the cell.

Various polynucleotide-based detection methods well known in the art maybe employed for the detection of PSCA-encoding nucleic acid moleculesand for the detection of PSCA expressing cells in a biological specimen.For example, RT-PCR methods may be used to selectively amplify a PSCAmRNA or fragment thereof, and such methods may be employed to identifycells expressing PSCA, as descried in Example 1 below. In a particularembodiment, RT-PCR is used to detect micrometastatic prostate cancercells or circulating prostate cancer cells. Various other amplificationtype detection methods, such as, for example, branched DNA methods, andvarious well known hybridization assays using DNA or RNA probes may alsobe used for the detection of PSCA-encoding polynucleotides or PSCAexpressing cells.

Various methods for the detection of proteins are well known in the artand may be employed for the detection of PSCA proteins and PSCAexpressing cells. To perform a diagnostic test based on proteins, asuitable protein sample is obtained and prepared using conventionaltechniques. Protein samples can be prepared, for example, simply byboiling a sample with SDS. The extracted protein can then be analyzed todetermine the presence of a PSCA protein using known methods. Forexample, the presence of specific sized or charged variants of a proteincan be identified using mobility in an electric filed. Alternatively,antibodies can be used for detection purposes. A skilled artisan canreadily adapt known protein analytical methods to determine if a samplecontains a PSCA protein.

Alternatively, PSCA expression can also be used in methods to identifyagents that decrease the level of expression of the PSCA gene. Forexample, cells or tissues expressing a PSCA protein can be contactedwith a test agent to determine the effects of the agent on PSCAexpression. Agents that activate PSCA expression can be used as anagonist of PSCA activity whereas agents that decrease PSCA expressioncan be used as an antagonist of PSCA activity.

PSCA PROMOTER AND OTHER EXPRESSION REGULATORY ELEMENTS

The invention further provides the expression control sequences found 5′of the of the newly identified PSCA gene in a form that can be used ingenerating expression vectors and transgenic animals. Specifically, thePSCA expression control elements, such as the PSCA promoter that canreadily be identified as being 5′ from the ATG start codon in the PSCAgene, can be used to direct the expression of an operably linked proteinencoding DNA sequence. Since PSCA expression is confined to prostatecells, the expression control elements are particularly useful indirecting the expression of an introduced transgene in a tissue specificfashion. A skilled artisan can readily use the PSCA gene promoter andother regulatory elements in expression vectors using methods known inthe art.

GENERATION OF TRANSGENIC ANIMAL

Another aspect of the invention provides transgenic non-human mammalscomprising PSCA nucleic acids. For example, in one application,PSCA-deficient non-human animals can be generated using standardknock-out procedures to inactivate a PSCA homologue or, if such animalsare non-viable, inducible PSCA homologue antisense molecules can be usedto regulate PSCA homologue activity/expression. Alternatively, an animalcan be altered so as to contain a human PSCA-encoding nucleic acidmolecule or an antisense-PSCA expression unit that directs theexpression of PSCA protein or the antisense molecule in a tissuespecific fashion. In such uses, a non-human mammal, for example a mouseor a rat is generated in which the expression of the PSCA homologue geneis altered by inactivation or activation and/or replaced by a human PSCAgene. This can be accomplished using a variety of art-known proceduressuch as targeted recombination. Once generated, the PSCA homologuedeficient animal, the animal that expresses PSCA (human or homologue) ina tissue specific manner, or an animal that expresses an antisensemolecule can be used to (I) identify biological and pathologicalprocesses mediated by the PSCA protein, (2) identify proteins and othergenes that interact with the PSCA proteins, (3) identify agents that canbe exogenously supplied to overcome a PSCA protein deficiency and (4)serve as an appropriate screen for identifying mutations within the PSCAgene that increase or decrease activity.

For example, it is possible to generate transgenic mice expressing thehuman minigene encoding PSCA in a tissue specific-fashion and test theeffect of over-expression of the protein in tissues and cells thatnormally do not contain the PSCA protein. This strategy has beensuccessfully used for other genes, namely bcl-2 (Veis et al. Cell 199375:229). Such an approach can readily be applied to the PSCAprotein/gene and can be used to address the issue of a potentialbeneficial or detrimental effect of the PSCA proteins in a specifictissue.

COMPOSITIONS

The invention provides a pharmaceutical composition comprising an PSCAnucleic acid molecule of the invention or an expression vector encodingan PSCA protein or encoding a fragment thereof and, optionally, asuitable carrier. The invention additionally provides a pharmaceuticalcomposition comprising an antibody or fragment thereof which recognizesand binds an PSCA protein. In one embodiment, the antibody or fragmentthereof is conjugated or linked to a therapeutic drug or a cytotoxicagent.

Suitable carriers for pharmaceutical compositions include any materialwhich when combined with the nucleic acid or other molecule of theinvention retains the molecule's activity and is non-reactive with thesubject's immune systems. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods. Such compositions may also beformulated within various lipid compositions, such as, for example,liposomes as well as in various polymeric compositions, such as polymermicrospheres.

The invention also provides a diagnostic composition comprising a PSCAnucleic acid molecule, a probe that specifically hybridizes to a nucleicacid molecule of the invention or to any part thereof, or an PSCAantibody or fragment thereof. The nucleic acid molecule, the probe orthe antibody or fragment thereof can be labeled with a detectablemarker. Examples of a detectable marker include, but are not limited to,a radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Further, theinvention provides a diagnostic composition comprising a PSCA-specificprimer pair capable of amplifying PSCA-encoding sequences usingpolymerase chain reaction methodologies, such as RT-PCR.

EXAMPLES Example 1 Identification and Molecular Characterization of aNovel prostrate-Specific Clel Surface Antigen (PSCA) MATERIALS ANDMETHODS

LAPC-4 Xenogafts: LAPC-4 xenografts were generated as described in Kleinet al, 1997, Nature Med. 3: 402-408.

RDA, Northern Analysis and RT-PCR: Representational difference analysisof androgen dependent and independent LAPC-4 tumors was performed aspreviously described (Braun et al., 1995, Mol. Cell. Biol. 15:4623-4630). Total RNA was isolated using Ultraspec® RNA isolationsystems (Biotecx, Houston, Tex.) according to the manufacturer'sinstructions. Northern filters were probed with a 660 bp RDA fragmentcorresponding to the coding sequence and part of the 3′ untranslatedsequence of PSCA or a ˜400 bp fragment of PSA. The human multiple tissueblot was obtained from Clontech and probed as specified. For reversetranscriptase (RT)-PCR analysis, first strand cDNA was synthesized fromtotal RNA using the GeneAmp RNA PCR core kit (Perkin Elmer-Roche, NewJersey). For RT-PCR of human PSCA transcripts, primers 5′-tgcttgccctgttgatggcag-(SEQ ID NO:12) and 3′-ccagagcagcaggccgagtgca-(SEQID NO:13) were used to amplify a ˜320 bp fragment. Thermal cycling wasperformed by 25-25 cycles of 95° for 30 sec, 60° for 30 sec and 72° for1 min, followed by extension at 72° for 10 min. Primers for GAPDH(Clontech) were used as controls. For mouse PSCA, the primers used were5′-ttctcctgctggccacctac-(SEQ ID NO:10) and 3′-gcagctcatcccttcacaat-(SEQID NO:11).

In Situ Hybridization Assay for PSCA mRNA: For mRNA in situhybridization, recombinant plasmid pCR II (1 ug, Invitrogen, San Diego,Calif.) containing the full-length PSCA gene was linearized to generatesense and antisense digoxigenin labeled riboprobes. In situhybridization was performed on an automated instrument (Ventana Gen II,Ventana Medical Systems) as previously described (Magi-Galluzzi et al.,1997, Lab. Invest. 76: 37-43). Prostate specimens were obtained from apreviously described database which has been expanded to ˜130 specimens(Magi-Galluzzi et al., supra). Slides were read and scored by twopathologists in a blinded fashion. Scores of 0-3 were assigned accordingto the percentage of positive cells (0=0%; 1=<25%; 2=25-50%; 3=>50%) andthe intensity of staining (0=0;1=1+; 2=2+; 3=3+). The two scores weremultiplied to give an overall score of 0-9.

RESULTS

Human PSCA cDNA: Representational Difference Analysis (RDA), a PCR-basedsubtractive hybridization technique, was used to compare gene expressionbetween hormone dependent and hormone independent variants of a humanprostate cancer xenograft (LAPC-4) and to isolate cDNAs upregulated inthe androgen-independent LAPC-4 subline. Multiple genes were cloned,sequenced, and checked for differential expression. One 660 bp fragment(clone #15) was identified which was found to be highly overexpressed inxenograft tumors when compared with normal prostate. Comparison of theexpression of this clone to that of PSA in normal prostate and xenografttumors suggested that clone #15 was relatively cancer specific (FIG. 9).

Sequence analysis revealed that clone #15 had no exact match in thedatabases, but shared 30% nucleotide homology with stem cell antigen 2,a member of the Thy-1/Ly-6 superfamily of glycosylphosphatidylinositol(GPI)-anchored cell surface antigens. Clone #15 encodes a 123 amino acidprotein which is 30% identical to SCA-2 (also called RIG-E) and containsa number of highly conserved cysteine residues characteristic of theLy-6/Thy-1 gene family (FIG. 3). Consistent with its homology to afamily of GPI-anchored proteins, clone #15 contains both anamino-terminal hydrophobic signal sequence and a carboxyl-terminalstretch of hydrophobic amino acids preceded by a group of small aminoacids defining a cleavage/binding site for GPI linkage (Udenfriend andKodukula, 1995, Ann. Rev. Biochem. 64: 563-591). It also contains fourpredicted N-glycosylation sites. Because of its strong homology to thestem cell antigen-2, clone #15 was renamed prostate stem cell antigen(PSCA). 5′ and 3′ PCR RACE analysis was then performed using cDNAobtained from the LAPC-4 androgen independent xenograft and the filllength cDNA nucleotide sequence (including the coding and untranslatedregions) was obtained. The nucleotide sequence of the full length cDNAencoding human PSCA is shown in FIG. 1A and the translated amino acidsequence is shown in FIG. 1B and in FIG. 3.

PSCA Expression is Prostate-Specific: The distribution of PSCA mRNA innormal human tissues was examined by Northern blot analysis. Theresults, shown in FIG. 9B, demonstrate that PSCA is expressedpredominantly in prostate, with a lower level of expression present inplacenta. Small amounts of mRNA can be detected in kidney and smallintestine after prolonged exposure and at approximately {fraction(1/100)}th of the level seen in prostate tissue. Reversetranscriptase-polymerase chain reaction (RT-PCR) analysis of PSCAexpression in normal human tissues also demonstrates that PSCAexpression is restricted. In a panel of normal tissues, high level PSCAmRNA expression was detected in prostate, with significant expressiondetected in placenta and tonsils (FIG. 7A). RT-PCR analysis of PSCAMnRNA expression in a variety of prostate cancer xenografts prostatecancer cell lines and other cell lines, and normal prostate showed highlevel expression restricted to normal prostate, the LAPC-4 and LAPC-9prostate cancer xenografts, and the ovarian cancer cell line A431 (FIG.7B).

The major PSCA transcript in normal prostate is ˜1 kb (FIG. 9B). MousePSCA expression was analyzed by RT-PCR in mouse spleen, liver, lung,prostate, kidney and testis. Like human PSCA, murine PSCA is expressedpredominantly in prostate. Expression can also be detected in kidney ata level similar to that seen for placenta in human tissues. These dataindicate that PSCA expression is largely prostate-specific.

The expression of PSCA, PSMA and PSA in prostate cancer cell lines andxenografts was compared by Northern blot analysis. The results shown inFIG. 10 demonstrate high level prostate cancer specific expression ofboth PSCA and PSMA, whereas PSA expression is not prostate cancerspecific.

PSCA is Expressed by a Subset of Basal Cells in Normal Prostate: Normalprostate contains two major epithelial cell populations-secretoryluminal cells and subjacent basal cells. In situ hybridizations wereperformed on multiple sections of normal prostate using an antisenseriboprobe specific for PSCA to localize its expression. As shown in FIG.11, PSCA is expressed exclusively in a subset of normal basal cells.Little to no staining is seen in stroma, secretory cells or infiltratinglymphocytes. Hybridization with sense PSCA riboprobes showed nobackground staining. Hybridization with an antisense probe for GAPDHconfirmed that the RNA in all cell types was intact. Because basal cellsrepresent the putative progenitor cells for the terminallydifferentiated secretory cells, these results suggest that PSCA may be aprostate-specific stem/progenitor cell marker (Bonkhoff et al., 1994,Prostate 24: 114-118). In addition, since basal cells areandrogen-independent, the association of PSCA with basal cells raisesthe possibility that PSCA may play a role in androgen-independentprostate cancer progression.

PSCA is Overexpressed in Prostate Cancer Cells: The initial analysiscomparing PSCA expression in normal prostate and LAPC-4 xenograft tumorssuggested that PSCA was overexpressed in prostate cancer. Asdemonstrated by the Northern blot analysis shown FIG. 9, LAPC-4 prostatecancer tumors strongly express PSCA; however, there is almost nodetectable expression in normal prostate. In contrast, PSA expression isclearly detectable in normal prostate, at levels 2-3 times those seen inthe LAPC-4 tumors. Thus, the expression of PSCA in prostate cancerappears to be the reverse of what is seen with PSA. While PSA isexpressed more strongly in normal than malignant prostate tissue, PSCAis expressed more highly in prostate cancer.

To confirm the prostate-specificity of PSCA expression, one hundredtwenty six paraffin-embedded prostate cancer specimens were analyzed bymRNA in situ hybridization for PSCA expression. Specimens were obtainedfrom primary tumors removed by radical prostatectomy or transurethralresection in all cases except one. All specimens were probed with both asense and antisense construct in order to control for backgroundstaining. Slides were assigned a composite score as describe underMaterials and Methods, with a score of 6 to 9 indicating strongexpression and a score of 4 meaning moderate expression. 102/126 (81%)of cancers stained strongly for PSCA, while another 9/126 (7%) displayedmoderate staining (FIGS. 11B and 11C). High grade prostaticintraepithelial neoplasia, the putative precursor lesion of invasiveprostate cancer, stained strongly positive for PSCA in 82% (97/118) ofspecimens (FIG. 3b) (Yang et al., 1997, Am. J. Path. 150: 693-703).Normal glands stained consistently weaker than malignant glands (FIG.11B). Nine specimens were obtained from patients treated prior tosurgery with hormone ablation therapy. Seven of nine (78%) of theseresidual presumably androgen-independent cancers overexpressed PSCA, apercentage similar to that seen in untreated cancers. Because such alarge percentage of specimens expressed PSCA mRNA, no statisticalcorrelation could be made between PSCA expression and pathologicalfeatures such as tumor stage and grade. These results suggest that PSCAmRNA overexpression is a common feature of androgen-dependent andindependent prostate cancer.

PSCA is Expressed in Androgen Receptor Negative Prostate Cancer CellLines: Although PSCA was initially cloned using subtractivehybridization, Northern blot analysis demonstrated strong PSCAexpression in both androgen-dependent and androgen-independent LAPC-4xenograft tumors (FIG. 9). Moreover, PSCA expression was detected in allprostate cancer xenografts and cell lines tested, including the LAPC-4cell line and xenograft, the LAPC-9 xenograft, and the LnCaP cell line(all androgen-dependent) as well as in the Al LAPC-4 xenograft subline,the LAPC-3 xenograft, the PC-3 and DU-145 cell lines, the AI LnCaPsubline, and the MatLyLu cell line (all androgen-independent).

PSCA expression in the androgen-independent, androgen receptor-negativeprostate cancer cell lines PC3 and DU145 was also detected byreverse-transcriptase polymerase chain reaction analysis. These datasuggest that PSCA can be expressed in the absence of functional androgenreceptor.

Example 2 Biochemical Characterization of PSCA MATERIALS AND METHODS

Polyclonal Antibodies and Immunoprecipitations: Rabbit polyclonalantiserum was generated against the syntheticpeptide—TARIRAVGLLTVISK—and affinity purified using a PSCA-glutathione Stransferase fusion protein. 293T cells were transiently transfected withpCDNA I (Invitrogen, San Diego, Calif.) expression vectors containingPSCA, CD59, E25 or vector alone by calcium phosphate precipitation.Immunoprecipitation was performed as previously described (Harlow andLane, 1988, Antibodies: A Laboratory Manual. (Cold Spring HarborPress)). Briefly, cells were labeled with 500 uCi of trans35S label(ICN, Irvine, Calif.) for six hours. Cell lysates and conditioned mediawere incubated with 1 ug of purified rabbit anti-PSCA antibody and 20 ulprotein A sepharose CLA4B (Pharmacia Biotech, Sweden) for two hours. Fordeglycosylation, immunoprecipitates were treated overnight at 37° with 1u N-glycosidase F (Boehringer Mannheim) or 0.1 u neuaminidase (Sigma, StLouis, Mo.) for 1 hour followed by overnight in 2.5 mU O-glycosidase(Boehringer Mannheim).

Flow Cytometry: For flow cytometric analysis of PSCA cell surfaceexpression, single cell suspensions were stained with 2 μg/ml ofpurified anti-PSCA antibody and a 1:500 dilution of fluoresceinisothiocyanate (FITC) labeled anti-rabbit IgG (Jackson Laboratories,West Grove, Pa.). Data was acquired on a FACScan (Becton Dickinson) andanalyzed using LYSIS II software. Control samples were stained withsecondary antibody alone. Glycosylphosphatidyl inositol linkage wasanalyzed by digestion of 2×10⁶ cells with 0.5 units ofphosphatidylinositol-specific phospholipase C (PI-PLC, BoehringerMannheim) for 90 min at 37° C. Cells were analyzed prior to and afterdigestion by either FACS scanning or immunoblotting.

RESULTS

PSCA is a GPI-Anchored Glycoprotein Expressed on the Cell Surface: Thededuced PSCA amino acid sequence predicts that PSCA is heavilyglycosylated and anchored to the cell surface through a GPI mechanism.In order to test these predictions, we produced an affinity purifiedpolyclonal antibody raised against a unique PSCA peptide (see Materialsand Methods) This peptide contains no glycosylation sites and waspredicted, based on comparison to the three dimensional structure ofCD59 (another GPI-anchored PSCA homologue), to lie in an exposed portionof the mature protein (Kiefer et al., 1994, Biochem. 33: 4471-4482).Recognition of PSCA by the affinity-purified antibody was demonstratedby immunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. The polyclonalantibody immunoprecipitates predominantly a 24 kd band fromPSCA-transfected, but not mock-transfected cells (FIG. 12A). Threesmaller bands are also present, the smallest being ˜10 kd. Theimmunoprecipitate was treated with N and O specific glycosidases inorder to determine if these bands represented glycosylated forms ofPSCA. N-glycosidase F deglycosylated PSCA, whereas O-glycosidase had noeffect (FIG. 12A). Some GPI-anchored proteins are known to have bothmembrane-bound and secreted forms (Fritz and Lowe, 1996, Am. J. Physiol.270: G176-G183). FIG. 12B indicates that some PSCA is secreted in the293T-overexpressing system. The secreted form of PSCA migrates at alower molecular weight than the cell surface-associated form, perhapsreflecting the absence of the covalent GPI-linkage. This result mayreflect the high level of expression in the 293T cell line and needs tobe confirmed in prostate cancer cell lines and in vivo.

Fluorescence activated cell sorting SACS) analysis was used to localizePSCA expression to the cell surface. Nonpermeabilized mock-transfected293T cells, PSCA-expressing 293T cells and LAPC-4 cells were stainedwith affinity purified antibody or secondary antibody alone. FIG. 12Cshows cell surface expression of PSCA in PSCA-transfected 293T andLAPC-4 cells, but not in mock-transfected cells. To confirm that thiscell surface expression is mediated by a covalent GPI-linkage, cellswere treated with GPI-specific phospholipase C (PLC). Release of PSCAfrom the cell surface by PLC was indicated by a greater than one logreduction in fluorescence intensity. Recovery of PSCA in post digestconditioned medium was also confirmed by immunoblotting. The specificityof phospholipase C digestion for GPI-anchored proteins was confirmed byperforming the same experiment on 293T cells transfected with theGPI-inked antigen CD59 or the non-GPI linked transmembrane protein E25a(Deleersnijder et al., 1996, J. Biol. Chem 271: 19475-19482). PLCdigestion reduced cell surface expression of CD59 to the same degree asPSCA but had no effect on E25. These results support the prediction thatPSCA is a glycosylated, GPI-anchored cell surface protein.

Example 3 Isolation of cDNA Encoding Murine PSCA Homologue

The human PSCA cDNA was used to search murine EST databases in order toidentify homologues for potential transgenic and knockout experiments.One EST obtained from fetal mouse and another from neonatal kidney were70% identical to the human cDNA at both the nucleotide and amino acidlevels. The homology between the mouse clones and human PSCA includedregions of divergence between human PSCA and its GPI-anchoredhomologues, indicating that these clones likely represented the mousehomologue of PSCA. Alignment of these ESTs and 5′ extension usingRACE-PCR provided the entire coding sequence (FIG. 2).

Example 4 Isolation of Human and Murine PSCA Genes MATERIALS AND METHODS

Genomic Cloning: Lambda phage clones containing the human PSCA gene wereobtained by screening a human genomic library (Stratagene) with a humanPSCA cDNA probe (Sambrook et al., 1989, Molecular Cloning (Cold SpringHarbor)). BAC (bacterial artificial chromosome) clones containing themurine PSCA gene were obtained by screening a murine BAC library (GenomeSystems, St. Louis, Mo.) with a murine PSCA cDNA probe. A 14 kb humanNot I fragment and a 10 kb murine Eco RI fragment were subcloned intopBluescript (Stratagene), sequenced, and restriction mapped.

Chromosome Mapping by Fluorescence In Situ Hybridization: Fluorescencein situ chromosomal analysis (FISH) was performed as previouslydescribed using overlapping human lambda phage clones (Rowley et al.,1990, Proc. Natl. Acad. Sci. USA 87: 9358-9362).

RESULTS

Structure of PSCA Gene: Human and murine genomic clones of approximately14 kb and 10 kb, respectively, were obtained and restriction mapped. Aschematic representation of the gene structures of human and murine PSCAand Ly6/Thy-1 is shown in FIG. 8. Both the human and murine genomicclones contain three exons encoding the translated and 3′ untranslatedregions of the PSCA gene. A fourth exon encoding a 5′ untranslatedregion is presumed to exist based on PSCA's homology to other members ofthe Ly-6 and Thy-1 gene families (FIG. 8).

Human PSCA Gene Maps to Chromosome 8q24.2: Southern blot analysis ofLAPC-4 genomic DNA revealed that PSCA is encoded by a single copy gene.Other Ly-6 gene family members contain four exons, including a firstexon encoding a 5′ untranslated region and three additional exonsencoding the translated and 3′ untranslated regions. Genomic clones ofhuman and murine PSCA containing all but the presumed 5′ first exon wereobtained by screening lambda phage libraries. Mouse and human PSCAclones had a similar genomic organization. The human clone was used tolocalize PSCA by fluorescence in situ hybridization analysis.Cohybridization of overlapping human PSCA lambda phage clones resultedin specific labeling only of chromosome 8 (FIG. 13). Ninety sevenpercent of detected signals localized to chromosome 8q24, of which 87%were specific for chromosome 8q24.2. These results show that PSCA islocated at chromosome 8, band q24.2.

Example 5 Generation of Monoclonal Antibodies Recognizing DifferentEpitopes of PSCA MATERIALS AND METHODS

A GST-PSCA fusion protein immunogen was used to raise antibodies in miceusing standard monoclonal antibody generation methodology. Briefly, thePSCA coding sequence corresponding to amino acids 18 through 98 of thehuman PSCA amino acid sequence shown in FIG. 1B was PCR-amplified usingthe primer pair:

5′- GGAGAATTCATGGCACTGCCCTGCTGTGCTAC (SEQ ID NO:14)

3′-GGAGAATTCCTAATGGGCCCCGCTGGCGTT (SEQ ID NO:15)

The amplified PSCA sequence was cloned into pGEX-2T (Pharmacia), used totransform E. coli, and the fusion protein isolated.

Flow cytometric analysis of cell surface PSCA expression was carried outon LAPC-9 mouse prostate cancer xenograft cells, the prostate cancercell line LAPC-4, or normal prostate epithelial cells (Clonetics) usingMAbs 3E6 and 1G8 and the mouse polyclonal serum described in Example 2.25,000 cells per sample were analyzed following staining with a 1 to 10dilution of either MAb 1G8, 3E6, or mouse polyclonal serum, followed bya 1 to 100 dilution of an FITC-labeled goat anti-mouse secondaryantibody. Background fluorescence (control) was determined by incubationof the samples with the secondary antibody only.

Epitope mapping of anti-PSCA monoclonal antibodies was conducted byWestern blot analysis of GST-PSCA fusion proteins. Briefly, 1 μgGST-PSCA fusion protein (amino acids 18-98) or a GST-PSCA amino terminalregion protein (N-terminal, amino acids 2-50), a GST-PSCA middle regionprotein (GST-middle, amino acids 46-109), or a GST-carboxyl terminalregion protein (GST-C-terminal, amino acids 85-123) were separated on a12% SDS-PAGE gel and transferred to nitrocellulose. The membrane wasprobed with a 1 to 250 dilution of concentrated tissue culturesupernatant of either 1G8 or 3E6 monoclonal antibody hybridomas and thenwith a peroxidase labeled secondary antibody and visualized by enhancedchemiluminesence.

RESULTS

Four hybridoma clones were selected and the tissue culture supernatantsevaluated by ELISA, FACS, Western blot, and immunoprecipitation. Theseanalyses indicated that two of the clones produce MAbs, designated 3E6and 1G8, which consistently recognize PSCA. Cell surface expressionanalysis of PSCA expression on cancerous and normal prostate epithelialcells by flow cytometry using MAbs 3E6 and 1G8 and the polyclonalantibody described in Example 2 is shown in FIG. 14.

PSCA MAbs 3E6 and 1G8 were epitope mapped by Western blot analysis ofGST-PSCA fusion proteins. 7- The results are shown in FIG. 15 andindicate that these MAbs recognize different epitopes on PSCA. MAb 3E6recognizes an epitope in the carboxy-terminal region of the protein,whereas MAb 1G8 recognizes an amino-terminal epitope (FIG. 15).

Example 6

This data provides epitope mapping of anti-PSCA monoclonal antibodies.

Monoclonal antibodies 1G8, 2H9, 3C5, and 4A10 recognize an epitoperesiding in the amino terminal region of the PSCA protein and monoclonalantibody 3E6 recognizes an epitope in the carboxyl-terminal region ofthe protein. GST-PSCA fusion proteins encoding either the amino terminalregion of the PSCA protein (N-terminal, amino acids 2-50), the middleregion (middle, amino acids 46-109), or the carboxyl terminal region(C-terminal amino acids 85-123) were used in an ELISA to identify theepitope recognized by 5 anti-PSCA monoclonal antibodies. 10 ng of theindicated fusion protein coated in wells of a microtiter plate wasincubated with either a 1:250 dilution of concentrated tissue culturesupernatants of hybridomas lG8 or 3E6 or with 1:10 dilutions ofsupernatants from hybridomas 2H9, 3C5, or 4A10. Binding of themonoclonal antibodies was detected by incubation with a 1:4,000 dilutionof peroxidase-labeled secondary antibody and developed with 3,3′5,5′tetramethylbenzidine base. Optical densities of the wells weredetermined at a wavelength of 450 nm. Data for IGg and 3E6 antibodiesrepresent the mean ±SD of triplicate determinations and data for 2H9,3C5, and 4A10 are the means ±the range of duplicate determinations.Strongest binding of the monoclonal antibodies to the various fusionproteins is indicated in bold. The results are in Table 1.

TABLE 1 1G8 3E6 2H9 3C5 4A10 N-terminal 1.262 ± 0.202 0.147 ± 0.0140.803 ± 0.033 2.230 ± 0.064 1.859 ± 0.071 Middle 0.588 ± 0.066 0.124 ±0.007 0.006 ± 0.010 0.002 ± 0.001 0.009 ± 0.002 C-terminal 0.088 ±0.025 >4.00 0.010 ± 0.010 0.066 ± 0.060 0.006 ± 0.003

Monoclonal antibodies 1G8, 2H9, 3C5, and 4A10 recognize an epitoperesiding in the amino terminal region of the PSCA protein and monoclonalantibody 3E6 recognizes an epitope in the carboxyl-terminal region ofthe protein. GST-PSCA fusion proteins encoding either the amino terminalregion of the PSCA protein (N-terminal, amino acids 2-50), the middleregion (middle, amino acids 46-109), or the carboxyl terminal region(C-terminal amino acids 85-123) were used in an ELISA to identify theepitope recognized by 5 anti-PSCA monoclonal antibodies. 10 ng of theindicated fusion protein coated in wells of a microtiter plate wasincubated with either a 1:250 dilution of concentrated tissue culturesupernatants of hybridomas 1G8 or3E6 or with 1:10dilutions ofsupernatants from hybridomas 2H5, 3C5, or4A10. Binding of the monoclonalantibodies was detected by incubation with a 1:4,000 dilution ofperoxidase-labeled secondary antibody and developed with 3,3′5,5′tetramethylbenzidine base. Optical densities of the wells weredetermined at a wavelength of 450 nm. Data for 1G8 and 3E6 antibodiesrepresent the mean ±SD of triplicate determinations and data for 2H5,3C5, and 4A10 are the means ±the range of duplicate determinations.Strongest binding of the monoclonal antibodies to the various fusionproteins is indicated in bold. These results are shown in Table 2.

TABLE 2 1G8 3E6 2H9 3C5 4A10 N-terminal 1.262 ± 0.202 0.147 ± 0.0140.803 ± 0.033 2.230 ± 0.064 1.859 ± 0.071 Middle 0.588 ± 0.066 0.124 ±0.007 0.006 ± 0.010 0.002 ± 0.001 0.009 ± 0.002 C-terminal 0.088 ±0.025 >4.00 0.010 ± 0.010 0.066 ± 0.060 0.006 ± 0.003

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1 <211>LENGTH: 998 <212> TYPE: DNA <213> ORGANISM: HUMAN PSCA (hPSCA) <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (543) <223> OTHERINFORMATION: any nucleotide (i.e. a, c, g or t) <221> NAME/KEY:misc_feature <222> LOCATION: (580) <223> OTHER INFORMATION: anynucleotide (i.e. a, c, g or t) <221> NAME/KEY: misc_feature <222>LOCATION: (584) <223> OTHER INFORMATION: any nucleotide (i.e. a, c, g ort) <221> NAME/KEY: misc_feature <222> LOCATION: (604) <223> OTHERINFORMATION: any nucleotide (i.e. a, c, g or t) <221> NAME/KEY:misc_feature <222> LOCATION: (608) <223> OTHER INFORMATION: anynucleotide (i.e. a, c, g or t) <221> NAME/KEY: misc_feature <222>LOCATION: (615) <223> OTHER INFORMATION: any nucleotide (i.e. a, c, g,or t) <221> NAME/KEY: misc_feature <222> LOCATION: (636) <223> OTHERINFORMATION: any nucleotide (i.e. a, c, g, or t) <221> NAME/KEY:misc_feature <222> LOCATION: (640) <223> OTHER INFORMATION: anynucleotide (i.e. a, c, g or t) <221> NAME/KEY: misc_feature <222>LOCATION: (646) <223> OTHER INFORMATION: any nucleotide (i.e. a, c, g,or t) <221> NAME/KEY: misc_feature <222> LOCATION: (697) <223> OTHERINFORMATION: any nucleotide (i.e. a, c, g or t) <221> NAME/KEY:misc_feature <222> LOCATION: (926) <223> OTHER INFORMATION: anynucleotide (i.e. a, c, g or t) <400> SEQUENCE: 1 agggagaggc agtgaccatgaaggctgtgc tgcttgccct gttgatggca ggcttggccc 60 tgcagccagg cactgccctgctgtgctact cctgcaaagc ccaggtgagc aacgaggact 120 gcctgcaggt ggagaactgcacccagctgg gggagcagtg ctggaccgcg cgcatccgcg 180 cagttggcct cctgaccgtcatcagcaaag gctgcagctt gaactgcgtg gatgactcac 240 aggactacta cgtgggcaagaagaacatca cgtgctgtga caccgacttg tgcaacgcca 300 gcggggccca tgccctgcagccggctgccg ccatccttgc gctgctccct gcactcggcc 360 tgctgctctg gggacccggccagctatagg ctctgggggg ccccgctgca gcccacactg 420 ggtgtggtgc cccaggcctttgtgccactc ctcacagaac ctggcccagt gggagcctgt 480 cctggttcct gaggcacatcctaacgcaag tttgaccatg tatgtttgca ccccttttcc 540 ccnaaccctg accttcccatgggccttttc caggattccn accnggcaga tcagttttag 600 tganacanat ccgcntgcagatggcccctc caaccntttn tgttgntgtt tccatggccc 660 agcattttcc acccttaaccctgtgttcag gcacttnttc ccccaggaag ccttccctgc 720 ccaccccatt tatgaattgagccaggtttg gtccgtggtg tcccccgcac ccagcagggg 780 acaggcaatc aggagggcccagtaaaggct gagatgaagt ggactgagta gaactggagg 840 acaagagttg acgtgagttcctgggagttt ccagagatgg ggcctggagg cctggaggaa 900 ggggccaggc ctcacatttgtggggntccc gaatggcagc ctgagcacag cgtaggccct 960 taataaacac ctgttggataagccaaaaaa aaaaaaaa 998 <210> SEQ ID NO 2 <211> LENGTH: 123 <212> TYPE:PRT <213> ORGANISM: HUMAN PSCA (hPSCA) <220> FEATURE: <221> NAME/KEY:SITE <222> LOCATION: (50)..(64) <221> NAME/KEY: SITE <222> LOCATION:(71)..(82) <400> SEQUENCE: 2 Met Lys Ala Val Leu Leu Ala Leu Leu Met AlaGly Leu Ala Leu Gln 1 5 10 15 Pro Gly Thr Ala Leu Leu Cys Tyr Ser CysLys Ala Gln Val Ser Asn 20 25 30 Glu Asp Cys Leu Gln Val Glu Asn Cys ThrGln Leu Gly Glu Gln Cys 35 40 45 Trp Thr Ala Arg Ile Arg Ala Val Gly LeuLeu Thr Val Ile Ser Lys 50 55 60 Gly Cys Ser Leu Asn Cys Val Asp Asp SerGln Asp Tyr Tyr Val Gly 65 70 75 80 Lys Lys Asn Ile Thr Cys Cys Asp ThrAsp Leu Cys Asn Ala Ser Gly 85 90 95 Ala His Ala Leu Gln Pro Ala Ala AlaIle Leu Ala Leu Leu Pro Ala 100 105 110 Leu Gly Leu Leu Leu Trp Gly ProGly Gln Leu 115 120 <210> SEQ ID NO 3 <211> LENGTH: 441 <212> TYPE: DNA<213> ORGANISM: MURINE PSCA (mPSCA) <400> SEQUENCE: 3 atgaagacagttttttttat cctgctggcc acctacttag ccctgcatcc aggtgctgct 60 ctgcagtgctattcatgcac agcacagatg aacaacagag actgtctgaa tgtacagaac 120 tgcagcctggaccagcacag ttgctttaca tcgcgcatcc gggccattgg actcgtgaca 180 gttatcagtaagggctgcag ctcacagtgt gaggatgact cggagaacta ctatttgggc 240 aagaagaacatcacgtgctg ctactctgac ctgtgcaatg tcaacggggc ccacaccctg 300 aagccacccaccaccctggg gctgctgacc gtgctctgca gcctgttgct gtggggctcc 360 agccgtctgtaggctctggg agagcctacc atagcccgat tgtgaaggga tgagctgcac 420 tccaccccacccccacacag g 441 <210> SEQ ID NO 4 <211> LENGTH: 123 <212> TYPE: PRT<213> ORGANISM: MURINE PSCA (mPSCA) <400> SEQUENCE: 4 Met Lys Thr ValPhe Phe Ile Leu Leu Ala Thr Tyr Leu Ala Leu His 1 5 10 15 Pro Gly AlaAla Leu Gln Cys Tyr Ser Cys Thr Ala Gln Met Asn Asn 20 25 30 Arg Asp CysLeu Asn Val Gln Asn Cys Ser Leu Asp Gln His Ser Cys 35 40 45 Phe Thr SerArg Ile Arg Ala Ile Gly Leu Val Thr Val Ile Ser Lys 50 55 60 Gly Cys SerSer Gln Cys Glu Asp Asp Ser Glu Asn Tyr Tyr Leu Gly 65 70 75 80 Lys LysAsn Ile Thr Cys Cys Tyr Ser Asp Leu Cys Asn Val Asn Gly 85 90 95 Ala HisThr Leu Lys Pro Pro Thr Thr Leu Gly Leu Leu Thr Val Leu 100 105 110 CysSer Leu Leu Leu Trp Gly Ser Ser Arg Leu 115 120 <210> SEQ ID NO 5 <211>LENGTH: 131 <212> TYPE: PRT <213> ORGANISM: HUMAN STEM CELL ANTIGEN-2(hSCA-2) <400> SEQUENCE: 5 Met Lys Ile Phe Leu Pro Val Leu Leu Ala AlaLeu Leu Gly Val Glu 1 5 10 15 Arg Ala Ser Ser Leu Met Cys Phe Ser CysLeu Asn Gln Lys Ser Asn 20 25 30 Leu Tyr Cys Leu Lys Pro Thr Ile Cys SerAsp Gln Asp Asn Tyr Cys 35 40 45 Val Thr Val Ser Ala Ser Ala Gly Ile GlyAsn Leu Val Thr Phe Gly 50 55 60 His Ser Leu Ser Lys Thr Cys Ser Pro AlaCys Pro Ile Pro Glu Gly 65 70 75 80 Val Asn Val Gly Val Ala Ser Met GlyIle Ser Cys Cys Gln Ser Phe 85 90 95 Leu Cys Asn Phe Ser Ala Ala Asp GlyGly Leu Arg Ala Ser Val Thr 100 105 110 Leu Leu Gly Ala Gly Leu Leu LeuSer Leu Leu Pro Ala Leu Leu Arg 115 120 125 Phe Gly Pro 130 <210> SEQ IDNO 6 <211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM: HUMAN PSCA(hPSCA) <400> SEQUENCE: 6 Met Lys Ala Val Leu Leu Ala Leu Leu Met AlaGly Leu Ala Leu Gln 1 5 10 15 Pro Gly Thr Ala Leu Leu Cys Tyr Ser CysLys Ala Gln Val Ser Asn 20 25 30 Glu Asp Cys Leu Gln Val Glu Asn Cys ThrGln Leu Gly Glu Gln Cys 35 40 45 Trp Thr Ala Arg Ile Arg Ala Val Gly LeuLeu Thr Val Ile Ser Lys 50 55 60 Gly Cys Ser Leu Asn Cys Val Asp Asp SerGln Asp Tyr Tyr Val Gly 65 70 75 80 Lys Lys Asn Ile Thr Cys Cys Asp ThrAsp Leu Cys Asn Ala Ser Gly 85 90 95 Ala His Ala Leu Gln Pro Ala Ala AlaIle Leu Ala Leu Leu Pro Ala 100 105 110 Leu Gly Leu Leu Leu Trp Gly ProGly Gln Leu 115 120 <210> SEQ ID NO 7 <211> LENGTH: 123 <212> TYPE: PRT<213> ORGANISM: MURINE PSCA (mPSCA) <400> SEQUENCE: 7 Met Lys Thr ValLeu Phe Leu Leu Leu Ala Thr Tyr Leu Ala Leu His 1 5 10 15 Pro Gly AlaAla Leu Gln Cys Tyr Ser Cys Thr Ala Gln Met Asn Asn 20 25 30 Arg Asp CysLeu Asn Val Gln Asn Cys Ser Leu Asp Gln His Ser Cys 35 40 45 Phe Thr SerArg Ile Arg Ala Ile Gly Leu Val Thr Val Ile Ser Lys 50 55 60 Gly Cys SerSer Gln Cys Glu Asp Asp Ser Glu Asn Tyr Tyr Leu Gly 65 70 75 80 Lys LysAsn Ile Thr Cys Cys Tyr Ser Asp Leu Cys Asn Val Asn Gly 85 90 95 Ala HisThr Leu Lys Pro Pro Thr Thr Leu Gly Leu Leu Thr Val Leu 100 105 110 CysSer Leu Leu Leu Trp Gly Ser Ser Arg Leu 115 120 <210> SEQ ID NO 8 <211>LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HUMAN PSCA (hPSCA) <400>SEQUENCE: 8 Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys1 5 10 15 <210> SEQ ID NO 9 <211> LENGTH: 12 <212> TYPE: PRT <213>ORGANISM: HUMAN PSCA (hPSCA) <400> SEQUENCE: 9 Val Asp Asp Ser Gln AspTyr Tyr Val Gly Lys Lys 1 5 10 <210> SEQ ID NO 10 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: RT-PCR PRIMER <400>SEQUENCE: 10 ttctcctgct ggccacctac 20 <210> SEQ ID NO 11 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: RT-PCRPRIMER <400> SEQUENCE: 11 gcagctcatc ccttcacaat 20 <210> SEQ ID NO 12<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: RT-PCR PRIMER <400> SEQUENCE: 12 tgcttgccct gttgatggca g 21<210> SEQ ID NO 13 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: RT-PCR PRIMER <400> SEQUENCE: 13 ccagagcagcaggccgagtg ca 22 <210> SEQ ID NO 14 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: RT-PCR PRIMER <400>SEQUENCE: 14 ggagaattca tggcactgcc ctgctgtgct ac 32 <210> SEQ ID NO 15<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: RT-PCR PRIMER <400> SEQUENCE: 15 ggagaattcc taatgggccccgctggcgtt 30

What is claimed is:
 1. A method of inhibiting the growth of prostatetumor cells expressing Prostate Stem Cell Antigen (PSCA), comprisingadministering to a patient a monoclonal antibody designated ATCC No.HB-12612, HB-12616, ATCC No. HB12618, or ATCC No. HB-12617 which bindsspecifically to the extracellular domain of PSCA in an amount effectiveto inhibit growth of the prostate tumor cells.
 2. The method of claim 1,wherein said antibody is conjugated to a cytotoxic agent.
 3. The methodof claim 1, wherein said antibody is conjugated to a radioisotope. 4.The method of claim 2, wherein said cytotoxic agent is selected from thegroup consisting of ricin, doxorubicin, daunorubicin taxol, ethiduimbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, and glucocorticoid.
 5. Amethod for selectively killing a cell expressing Prostate Stem CellAntigen (PSCA) comprising reacting a monoclonal antibody designated ATCCNo. HB-12612, ATCC No. HB-12616, ATCC No. HB12618, or ATCC No. HB-12617conjugated to a therapeutic agent with the cell so that the therapeuticagent so conjugated can kill the cell expressing PSCA.